The thyroid hormones are present in blood mainly in a protein-bound form. The most important carrier protein for this is thyroxine-binding globulin, which is denoted TBG, to which ca. 80% of the total thyroxine and triiodothyronine present in blood is bound. Approximately 5 to 10% of the total thyroxine present is bound to albumin and about 10 to 15% of the total thyroxine present is bound to pre-albumin. The totality of these proteins is also referred to as thyroxine-binding protein (TBP). Only very small proportions of thyroxine and triiodothyronine is present in blood in a free form; usually ca. 0.03% of T.sub.4 or 0.3% of T.sub.3. Only the released hormones are physiologically active whereas the bound hormones T.sub.3 and T.sub.4 circulate in the blood as a metabolically inert transport form and serve as a buffer to regulate the level. It is therefore important to determine the total thyroxine content as well as the proportion of free thyroxine and free triiodothyronine in blood since this represents the functional thyroid status independent of changes in the concentration or saturation of the thyroxine binding protein.
Various methods are known for the determination of total thyroxine (T.sub.4), free thyroxine (FT.sub.4) and triiodothyronine (T.sub.3). A difficulty in all of these known methods is the provision of a standard which is suitable for the calibration.
For the calibration it is necessary to produce human serum standards with particular thyroxine or triiodothyronine concentrations and a defined FT.sub.4 or FT.sub.3 content. Human serum has previously been used as starting material for this. Firstly all the thyroxine and triiodothyronine in a free as well as in a bound form must be removed from the human serum and then the human serum treated in this way is supplemented with known levels of thyroxine or triiodothyronine. Various methods are described in the literature for this purpose such as treatment with active charcoal, with ion exchangers or with carrier-bound anti-T.sub.4 or anti-T.sub.3 antibodies. These methods are all very complicated. A further problem is that each human serum sample has a different composition thus leading to large variations in the individual lots and as a consequence the reproducibility leaves much to be desired.
A further disadvantage of these known standard solutions for the determination of T.sub.3 and T.sub.4 is that these solutions, containing human serum, cannot be stored for longer periods since, on the one hand, the protein which is present is gradually denatured and on the other hand there is a shift in the set equilibrium between bound and free T.sub.3 and T.sub.4.
It is known from EP-A 0 337 467 that in order to solve the above-mentioned problems, a standard solution can be used to determine thyroxine or triiodothyronine levels which is still stable after long periods of time and can be produced in a simple manner using relatively cheap starting materials by using at least one standard solution for the calibration that contains TBG and thyroxine or triiodothyronine dissolved in a buffer solution.
It was established that, in a solution containing TBG and thyroxine or triiodothyronine dissolved in a buffer system, the equilibrium which establishes between bound and free T.sub.4 or T.sub.3 is just as in human serum. Surprisingly, the incomplete equilibration system according to the present invention, in which components of the natural thyroxine-binding proteins are missing, yields even better comparative values than a natural matrix based on human serum which was first made free from and then augmented with thyroid hormone, so that after appropriate calibration such solutions can be used as standard solutions. Thus, in accordance with the present invention a standard solution is provided for a process to determine thyroxine or triiodothyronine which is simple to produce.
Accordingly, it was an object of the present invention to provide a standard solution for a process to determine thyroxine or triiodothyronine which is also stable after longer storage and which can be produced in a simple manner using cheap raw materials.
This object was achieved by a process for the determination of thyroxine or triiodothyronine in serum wherein at least one standard solution is used for calibration containing TBG and thyroxine or triiodothyronine respectively dissolved in a buffer solution.
It has also been found that a further quite considerable improvement of the stability on storage can be achieved if TBG from bovine serum is used in the aforementioned methods.